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Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins
Published on: July 1, 2011
DIRECT AND INDIRECT FLUORESCENT-ANTIBODY TECHNIQUES FOR THE PSITTACOSIS-LYMPHOGRANULOMA VENEREUM-TRACHOMA GROUP OF
Abstract:
Ross, Martin R. (Connecticut State Department of Health, Hartford) and Earle K. Borman. Direct and indirect fluorescent-antibody techniques for the psittacosis-lymphogranuloma venereum-trachoma group of agents. J. Bacteriol. 85:851-858. 1963.-Direct and indirect fluorescent-antibody (FA) techniques were developed for the detection of group antigen in infected tissue cultures and the titration of group antibody in human antiserum. The growth of the agent of meningopneumonitis (MP) in mouse embryo lung cell monolayers was followed by infectivity and complement-fixing (CF) antigen titrations, and cytological examination of FA stained cultures. Although infectivity and CF antigen reached a peak at 2 days and remained constant for an additional 3 days, only cells tested 2 to 3 days after infection were suitable for FA staining with labeled anti-MP serum because of excessive artifacts in the older cultures. Fluorescein isothiocyanate-labeled rooster and guinea pig anti-MP serums and human antipsittacosis serums were titrated in direct FA and hemagglutination-inhibition (HI) tests. The rooster conjugate showed brighter staining and higher antibody titers than the guinea pig or human conjugates and was more effective in detecting minimal amounts of virus antigen. FA staining reactions with 1 and 2 units of labeled rooster serum were inhibited by unlabeled rooster serum but clear-cut inhibition with human antipsittacosis serum could not be demonstrated. The indirect FA technique was successfully used for the titration of group antibody in human serum. A comparison of the indirect FA, HI, and CF tests showed the indirect FA technique to be intermediate in sensitivity between the HI and CF tests. None of the three tests showed significant cross reactions with human serums reactive for influenza A and B; parainfluenza 1, 2, and 3; respiratory syncytial virus; Q fever; or the primary atypical pneumonia agent.
Insights
Direct and indirect fluorescent-antibody (FA) techniques were developed to detect psittacosis-lymphogranuloma venereum-trachoma group antigen and antibody. The indirect FA test showed intermediate sensitivity compared to hemagglutination-inhibition and complement-fixing tests.
Area of Science:
- Microbiology
- Immunology
- Virology
Background:
- The psittacosis-lymphogranuloma venereum-trachoma (PLT) group of agents are significant human pathogens.
- Accurate diagnostic methods are crucial for identifying infections caused by these agents.
Purpose of the Study:
- To develop and evaluate direct and indirect fluorescent-antibody (FA) techniques for detecting PLT group antigen and antibody.
- To compare the sensitivity of the indirect FA test with other serological assays.
Main Methods:
- Development of direct and indirect FA techniques using fluorescein isothiocyanate-labeled antisera.
- Infection of mouse embryo lung cell monolayers with the agent of meningopneumonitis (MP).
- Titration of antibodies using direct FA, indirect FA, hemagglutination-inhibition (HI), and complement-fixing (CF) tests.
Main Results:
- Direct FA staining was effective for detecting group antigen in infected cell cultures, with optimal staining observed 2-3 days post-infection.
- Rooster anti-MP conjugate demonstrated superior staining intensity and higher antibody titers compared to guinea pig or human conjugates.
- The indirect FA technique successfully titrated group antibody in human serum and showed intermediate sensitivity between HI and CF tests.
- No significant cross-reactivity was observed with human sera positive for other respiratory viruses or Q fever.
Conclusions:
- Direct and indirect FA techniques are valuable tools for the diagnosis of PLT group infections.
- The indirect FA test offers a sensitive method for antibody detection, with performance characteristics between HI and CF assays.
- These FA techniques demonstrate specificity and can aid in differentiating PLT group infections from other viral and rickettsial infections.
